Autocrine signaling involved in cell volume regulation: the role of released transmitters and plasma membrane receptors

Cell volume regulation is a basic homeostatic mechanism transcendental for the normal physiology and function of cells. It is mediated principally by the activation of osmolyte transport pathways that result in net changes in solute concentration that counteract cell volume challenges in its constancy. This process has been described to be regulated by a complex assortment of intracellular signal transduction cascades. Recently, several studies have demonstrated that alterations in cell volume induce the release of a wide variety of transmitters including hormones, ATP and neurotransmitters, which have been proposed to act as extracellular signals that regulate the activation of cell volume regulatory mechanisms. In addition, changes in cell volume have also been reported to activate plasma membrane receptors (including tyrosine kinase receptors, G-protein coupled receptors and integrins) that have been demonstrated to participate in the regulatory process of cell volume. In this review, we summarize recent studies about the role of changes in cell volume in the regulation of transmitter release as well as in the activation of plasma membrane receptors and their further implications in the regulation of the signaling machinery that regulates the activation of osmolyte flux pathways. We propose that the autocrine regulation of Ca2+-dependent and tyrosine phosphorylation-dependent signaling pathways by the activation of plasma membrane receptors and swelling-induced transmitter release is necessary for the activation/regulation of osmolyte efflux pathways and cell volume recovery. Furthermore, we emphasize the importance of studying these extrinsic signals because of their significance in the understanding of the physiology of cell volume regulation and its role in cell biology in vivo, where the constraint of the extracellular space might enhance the autocrine or even paracrine signaling induced by these released transmitters.     

Regulation of the Cellular Content of the Organic Osmolyte Taurine in Mammalian Cells

Change in the intracellular concentration of osmolytes or the extracellular tonicity results in a rapid transmembrane water flow in mammalian cells until intracellular and extracellular tonicities are equilibrated. Most cells respond to the osmotic cell swelling by activation of volume-sensitive flux pathways for ions and organic osmolytes to restore their original cell volume. Taurine is an important organic osmolyte in mammalian cells, and taurine release via a volume-sensitive taurine efflux pathway is increased and the active taurine uptake via the taurine specific taurine transporter TauT decreased following osmotic cell swelling. The cellular signaling cascades, the second messengers profile, the activation of specific transporters, and the subsequent time course for the readjustment of the cellular content of osmolytes and volume vary from cell type to cell type. Using Ehrlich ascites tumor cells, NIH3T3 mouse fibroblasts and HeLa cells as biological systems, it is revealed that phospholipase A₂-mediated mobilization of arachidonic acid from phospholipids and subsequent oxidation of the fatty acid via lipoxygenase systems to potent eicosanoids are essential elements in the signaling cascade that is activated by cell swelling and leads to release of osmolytes. The cellular signaling cascade and the activity of the volume-sensitive taurine efflux pathway are modulated by elements of the cytoskeleton, protein tyrosine kinases/phosphatases, GTP-binding proteins, Ca²⁺/calmodulin, and reactive oxygen species and nucleotides. Serine/threonine phosphorylation of the active taurine uptake system TauT or a putative regulator, as well as change in the membrane potential, are important elements in the regulation of TauT activity. A model describing the cellular sequence, which is activated by cell swelling and leads to activation of the volume-sensitive efflux pathway, is presented at the end of the review.     

Taurine and cell volume maintenance in the shark rectal gland: cellular fluxes and kinetics

Tissue slices of shark rectal gland are studied to examine the kinetics of the cellular fluxes of taurine, a major intracellular osmolyte in this organ. Maintenance of high steady-state cell taurine (50 mM) is achieved by a ouabain-sensitive active Na+-dependent uptake process and a relatively slow efflux. Uptake kinetics are described by two saturable taurine transport components (high-affinity, Km 60 microM; and low-affinity, Km 9 mM). [14C]Taurine uptake is enhanced by external Cl-, inhibited by beta-alanine and unaffected by inhibitors of the Na+/K+/2Cl- co-transport system. Two cellular efflux components of taurine are documented. Incubation of slices in p-chloromercuribenzene sulfonate (1 mM) reduces taurine uptake, increases efflux of taurine and induces cell swelling. Studies of efflux in isotonic media with various cation and anion substitutions demonstrate that high-K+ markedly enhances taurine efflux irrespective of cell volume changes (i.e. membrane stretching is not involved). Moreover, iso-osmotic cell swelling induced in media containing propionate is not associated with enhanced efflux of taurine from the cells. It is suggested that external K+ exerts a specific effect on the cytoplasmic membrane to increase its permeability to taurine.     

Regulation of intracellular glycine as an organic osmolyte in early preimplantation mouse embryos

GLYT1, a glycine transporter belonging to the neurotransmitter transporter family, has recently been identified as a novel cell volume-regulatory mechanism in the earliest stages of the mouse preimplantation embryo. It apparently acts by regulating the steady-state intracellular concentration of glycine, which functions as an organic osmolyte in embryos, to balance external osmolarity and thus maintain cell volume. GLYT1 in embryos was the first mammalian organic osmolyte transporter identified that appears to function in cell volume control under conditions of normal osmolarity, rather than being a response to the stress of chronic hypertonicity. Its maximal rate of transport was shown to be regulated by osmolarity. However, it was not known whether this osmotic regulation of the rate of glycine transport is sufficient to account for the observed control of steady-state intracellular glycine levels as a function of osmolarity in embryos. Here, we show that the intracellular accumulation of glycine in embryos is a direct function of the rate of glycine uptake via GLYT1. In addition, we have shown that the rate of efflux, likely via the volume-regulated anion and organic osmolyte channel in embryos, is also under osmotic regulation and contributes substantially to the control of steady-state glycine concentrations. Together, control of both the rate of uptake and rate of efflux of glycine underlies the mechanism of osmotic regulation of the steady-state concentration of glycine and hence cell volume in early embryos.     

Mechanism of thiyl radical-catalyzed isomerization of unsaturated fatty acid residues in homogeneous solution and in liposomes

Taurine is an important osmolyte involved in cell volume regulation. During regulatory volume decrease it is released via a volume-sensitive organic osmolyte/anion channel. Several molecules have been suggested as candidates for osmolyte release. In this study, we chose three of these, namely ClC-2, ClC-3 and ICln, because of their expression in rat astrocytes, a cell type which is known to release taurine under hypotonic stress, and their activation by hypotonic shock. As all three candidates were also suggested to be chloride channels, we investigated their permeability for both chloride and taurine under isotonic and hypotonic conditions using the Xenopus laevis oocyte expression system. We found a volume-sensitive increase of chloride permeability in ClC-2-expressing oocytes only. Yet, the taurine permeability was significantly increased under hypotonic conditions in oocytes expressing any of the tested candidates. Further experiments confirmed that the detected taurine efflux does not represent unspecific leakage. These results suggest that ClC-2, ClC-3 and ICln either participate in taurine transport themselves or upregulate an endogenous oocyte osmolyte channel. In either case, the taurine efflux of oocytes not being accompanied by an increased chloride flux suggests that taurine and chloride can be released via two separate pathways.     

Osmolyte channel regulation by ionic strength in skate RBC

The aim of this study was to determine whether the opening of the osmolyte channel in skate red blood cells (RBC) is regulated by intracellular electrolyte concentration and conductivity. Consistent with previous studies, experiments with hyperosmotic preincubation before cell swelling or swelling with an isosmotic electrolyte (e.g., ammonium chloride) showed that an increase in ionic strength inhibits the opening of the taurine channel. However, a decrease in intracellular ionic strength did not always stimulate taurine efflux to the same degree. Whereas hyposmotic swelling caused a large increase in taurine efflux, swelling induced by treatment with isosmotic nonelectrolytes produced much smaller stimulation. Results with assays for band 3 phosphorylating enzymes were consistent with those from the taurine efflux studies; stimulation of enzyme activity was lower in cells that were swollen with isosmotic nonelectrolyte media than in cells swollen in hyposmotic media. These results indicate that a decrease in ionic strength is not the only signal for the opening of the taurine channel in skate RBC. Ionic strength does affect channel activity, but there must also be some other regulator.     

Characterization of volume-sensitive organic osmolyte efflux and anion current in Xenopus oocytes

Cell swelling activates an outwardly rectifying anion current in numerous mammalian cell types. An extensive body of evidence indicates that the channel responsible for this current is the major pathway for volume regulatory organic osmolyte loss. Cell swelling also activates an outwardly rectifying anion current in Xenopus oocytes. Unlike mammalian cells, oocytes allow the direct study of both swelling-activated anion current and organic osmolyte efflux under nearly identical experimental conditions. We therefore exploited the unique properties of oocytes in order to examine further the relationship between anion channel activity and swelling-activated organic osmolyte transport. Swelling-activated anion current and organic osmolyte efflux were studied in parallel in batches of oocytes obtained from single frogs. The magnitude of swelling-activated anion current and organic osmolyte efflux exhibited a positive linear correlation. In addition, the two processes had similar pharmacological characteristics and activation, rundown and reactivation kinetics. The present study provides further strong support for the concept that the channel responsible for swelling-activated Cl⁻ efflux and the outwardly rectifying anion conductance is also the major pathway by which organic osmolytes are lost from vertebrate cells during regulatory volume decrease. Received: 22 April 1996/Revised: 18 December 1996     

On the role of G-protein coupled receptors in cell volume regulation.

Cell volume is determined genetically for each cell lineage, but it is not a static feature of the cell. Intracellular volume is continuously challenged by metabolic reactions, uptake of nutrients, intracellular displacement of molecules and organelles and generation of ionic gradients. Moreover, recent evidence raises the intriguing possibility that changes in cell volume act as signals for basic cell functions such as proliferation, migration, secretion and apoptosis. Cells adapt to volume increase by a complex, dynamic process resulting from the concerted action of volume sensing mechanisms and intricate signaling chains, directed to initiate the multiple adaptations demanded by a change in cell volume, among others adhesion reactions, membrane and cytoskeleton remodeling, and activation of the osmolyte pathways leading to reestablish the water balance between extracellular/intracellular or intracellular/intracellular compartments. In multicellular organisms, a continuous interaction with the external milieu is fundamental for the dynamics of the cell. It is in this sense that the recent surge of interest about the influence on cell volume control by the most extended family of signaling elements, the G proteins, acquires particular importance. As here reviewed, a large variety of G-protein coupled receptors (GPCRs) are involved in this interplay with cell volume regulatory mechanisms, which amplifies and diversifies the volume-elicited signaling chains, providing a variety of routes towards the multiple effectors related to cell volume changes.     

Na(+)/H(+) exchanger NHE1 as plasma membrane scaffold in the assembly of signaling complexes

The plasma membrane Na⁺/H⁺ exchanger NHE1 has an established function in intracellular pH and cell volume homeostasis by catalyzing electroneutral influx of extracellular Na⁺ and efflux of intracellular H⁺. A second function of NHE1 as a structural anchor for actin filaments through its direct binding of the ezrin, radixin, and moesin (ERM) family of actin-binding proteins was recently identified. ERM protein binding and actin anchoring by NHE1 are necessary to retain the localization of NHE1 in specialized plasma membrane domains and to promote cytoskeleton-dependent processes, including actin filament bundling and cell-substrate adhesions. This review explores a third function of NHE1, as a plasma membrane scaffold in the assembly of signaling complexes. Through its coordinate functions in H⁺ efflux, actin anchoring, and scaffolding, we propose that NHE1 promotes protein interactions and activities, assembles signaling complexes in specialized plasma membrane domains, and coordinates divergent signaling pathways. hydrogen ion efflux; intracellular pH; molecular scaffold     

Mitogen-activated protein kinases as key players in osmotic stress signaling

BackgroundOsmotic stress arises from the difference between intracellular and extracellular osmolality. It induces cell swelling or shrinkage as a consequence of water influx or efflux, which threatens cellular activities. Mitogen-activated protein kinases (MAPKs) play central roles in signaling pathways in osmotic stress responses, including the regulation of intracellular levels of inorganic ions and organic osmolytes.Scope of reviewThe present review summarizes the cellular osmotic stress response and the function and regulation of the vertebrate MAPK signaling pathways involved. We also describe recent findings regarding apoptosis signal-regulating kinase 3 (ASK3), a MAP3K member, to demonstrate its regulatory effects on signaling molecules beyond MAPKs.Major conclusionsMAPKs are rapidly activated by osmotic stress and have diverse roles, such as cell volume regulation, gene expression, and cell survival/death. There is significant cell type specificity in the function and regulation of MAPKs. Based on its activity change during osmotic stress and its regulation of the WNK1-SPAK/OSR1 pathway, ASK3 is expected to play important roles in osmosensing mechanisms and cellular functions related to osmoregulation.General significanceMAPKs are essential for various cellular responses to osmotic stress; thus, the identification of the upstream regulators of MAPK pathways will provide valuable clues regarding the cellular osmosensing mechanism, which remains elusive in mammals. The elucidation of in vivo MAPK functions is also important because osmotic stress in physiological and pathophysiological conditions often results from changes in the intracellular osmolality. These studies potentially contribute to the establishment of therapeutic strategies against diseases that accompany osmotic perturbation.     

The extracellular calcium-sensing receptor and cell-cell signaling in epithelia

In multicellular organisms, cells are crowded together in organized communities, surrounded by an interstitial fluid of extremely limited volume. Local communication between adjacent cells is known to occur through gap junctions in cells that are physically connected, or through the release of paracrine signaling molecules (e.g. ATP, glutamate, nitric oxide) that diffuse to their target receptors through the extracellular microenvironment. Recent evidence hints that calcium ions may possibly be added to the list of paracrine messengers that allow cells to communicate with one another. Local fluctuations in extracellular [Ca2+] can be generated as a consequence of intracellular Ca2+ signaling events, owing to the activation of Ca2+ influx and efflux pathways at the plasma membrane. In intact tissues, where the interstitial volumes between cells are much smaller than the cells themselves, this can result in significant alterations in external [Ca2+]. This article will explore emerging evidence that these extracellular [Ca2+] changes can be detected by the extracellular calcium-sensing receptor (CaR) on adjacent cells, forming the basis for a paracrine signaling system. Such a mechanism could potentially provide CaR-expressing cells with the means to sense the Ca2+ signaling status of their neighbors, and expand the utility of the intracellular Ca2+ signal to a domain outside the cell.     

Regulation of cell volume via microvillar ion channels

A novel mechanism of cellular volume regulation is presented, which ensues from the recently introduced concept of transport and ion channel regulation via microvillar structures (Lange K, 1999, J Cell Physiol 180:19-35). According to this notion, the activity of ion channels and transporter proteins located on microvilli of differentiated cells is regulated by changes in the structural organization of the bundle of actin filaments in the microvillar shaft region. Cells with microvillar surfaces represent two-compartment systems consisting of the cytoplasm on the one side and the sum of the microvillar tip (or, entrance) compartments on the other side. The two compartments are separated by the microvillar actin filament bundle acting as diffusion barrier ions and other solutes. The specific organization of ion and water channels on the surface of microvillar cell types enables this two-compartment system to respond to hypo- and hyperosmotic conditions by activation of ionic fluxes along electrochemical gradients. Hypotonic exposure results in swelling of the cytoplasmic compartment accompanied by a corresponding reduction in the length of the microvillar diffusion barrier, allowing osmolyte efflux and regulatory volume decrease (RVD). Hypertonic conditions, which cause shortening of the diffusion barrier via swelling of the entrance compartment, allow osmolyte influx for regulatory volume increase (RVI). Swelling of either the cytoplasmic or the entrance compartment, by using membrane portions of the microvillar shafts for surface enlargement, activates ion fluxes between the cytoplasm and the entrance compartment by shortening of microvilli. The pool of available membrane lipids used for cell swelling, which is proportional to length and number of microvilli per cell, represents the sensor system that directly translates surface enlargements into activation of ion channels. Thus, the use of additional membrane components for osmotic swelling or other types of surface-expanding shape changes (such as the volume-invariant cell spreading or stretching) directly regulates influx and efflux activities of microvillar ion channels. The proposed mechanism of ion flux regulation also applies to the physiological main functions of epithelial cells and the auxiliary action of swelling-induced ATP release. Furthermore, the microvillar entrance compartment, as a finely dispersed ion-accessible peripheral space, represents a cellular sensor for environmental ionic/osmotic conditions able to detect concentration gradients with high lateral resolution. Volume regulation via microvillar surfaces is only one special aspect of the general property of mechanosensitivity of microvillar ionic pathways.     

Hypo-osmotic swelling-activated release of organic osmolytes in brain slices: implications for brain oedema in vivo

A decrease in the intracellular levels of osmotically active species has invariably been seen after swelling of mammalian brain tissue preparations. The exact identity of the species, and the manner of their decrease, remain to be described. We investigated the swelling-activated decrease of organic osmolytes in rat cortical brain slices using (1)H- and (31)P-magnetic resonance spectroscopy. We found that acute hypo-osmotic shock causes decreases in the levels of a range of intracellular amino acids and amino acid derivatives, N-acetyl-aspartate, creatine, GABA, glutamate, hypotaurine, and also in the levels of the methylamines glycerol-phosphorylcholine, phosphorylcholine and choline. Incubation of cortical slices with the anion channel blockers niflumic acid and tamoxifen caused inhibition of organic osmolyte efflux, suggesting that such osmolyte efflux occurs through anion channels. Intracellular phosphocreatine was also seen to decrease during acute hypo-osmotic superfusion, although intracellular ATP remained constant. In addition, the acidification of an intracellular compartment was observed during hypo-osmotic superfusion. Our results suggest a link between brain energy reserve and brain osmoregulation.     

Purinergic-independent calcium signaling mediates recovery from hepatocellular swelling: implications for volume regulation.

Swelling of hepatocytes and other epithelia activates volume-sensitive ion channels that facilitate fluid and electrolyte efflux to restore cell volume, but the responsible signaling pathways are incompletely defined. Previous work in model HTC rat hepatoma cells has indicated that swelling elicits ATP release, which stimulates P2 receptors and activates Cl(-) channels, and that this mechanism is essential for hepatocellular volume recovery. Since P2 receptors are generally coupled to Ca(2+) signaling pathways, we determined whether hepatocellular swelling affected cytosolic [Ca(2+)], and if this involved a purinergic mechanism. Exposure of HTC cells to hypotonic media evoked an increase in cytosolic [Ca(2+)], which was followed by activation of K(+) and Cl(-) currents. Maneuvers that interfered with swelling-induced increases in cytosolic [Ca(2+)], including extracellular Ca(2+) removal and intracellular Ca(2+) store depletion with thapsigargin, inhibited activation of membrane currents and volume recovery. However, the swelling-induced increases in cytosolic [Ca(2+)] were unaffected by either extracellular ATP depletion with apyrase or blockade of P2 receptors with suramin. These findings indicate that swelling elicits an increase in hepatocellular Ca(2+), which is essential for ion channel activation and volume recovery, but that this increase does not stem from activation of volume-sensitive P2 receptors. Collectively, these observations imply that regulatory responses to hepatocellular swelling involve a dual requirement for a purinergic-independent Ca(2+) signaling cascade and a Ca(2+)-independent purinergic signaling pathway.     

The existence of molecular water pumps in the nervous system: a review of the evidence

Recently, the presence if both influx and efflux molecular water pumps (MWP's) in vertebrate cells has been reported. These appear to use a common mechanism; the intercompartmental cotransport of water uphill against a gradient as a hydrophylic osmolyte is transported down its own gradient, in a regulated fashion, by a membrane spanning cotransporter protein. In each case, the dwell time of the transported osmolyte is short in that it is metabolically converted and its products either eliminated or recycled, thereby maintaining the required high intercompartmental gradient. An influx water pump osmolyte has been identified as a sodium-glucose complex, and an efflux water pump osmolyte as N-acetylhistidine. These osmolytes may also be archetypal representatives of many other osmolytes with similar functions in a variety of cells. When recycled, the osmolyte metabolites appear to be dewatered during high affinity binding that is associated with their active transport back across the membrane prior to intracellular resynthesis of the osmolyte. Since these cyclical systems result in the pumping of water, they also appear to create a previously unrecognized motive force which results in the establishment of unidirectional transcellular water flows between apical and basolateral cell membranes. As neurons represent highly specialized forms of animal cells, and cells which are also extremely sensitive to changes in osmotic pressure, the presence of these water pumps in the CNS could be significant. There would be connotations with regard to how neurons regulate water balance and transaxonal flow as well as to how these factors affect the integrated function of the nervous system. In this article, evidence of the presence of MWP's in the nervous system, and how they might relate to aspects of both normal and abnormal brain function is reviewed.     

Regulation of mitochondrial matrix volume

Mitochondrial volume homeostasis is a housekeeping cellular function essential for maintaining the structural integrity of the organelle. Changes in mitochondrial volume have been associated with a wide range of important biological functions and pathologies. Mitochondrial matrix volume is controlled by osmotic balance between cytosol and mitochondria. Any dysbalance in the fluxes of the main intracellular ion, potassium, will thus affect the osmotic balance between cytosol and the matrix and promote the water movement between these two compartments. It has been hypothesized that activity of potassium efflux pathways exceeds the potassium influx in functioning mitochondria and that potassium concentration in matrix could be actually lower than in cytoplasm. This hypothesis provides a clear-cut explanation for the mitochondrial swelling observed after mitochondrial depolarization, mitochondrial calcium overload, or opening of permeability transition pore. It should also be noted that the rate of water flux into or out of the mitochondrion is determined not only by the osmotic gradient that acts as the driving force for water transport but also by the water permeability of the inner membrane. Recent data suggest that the mitochondrial inner membrane has also specific water channels, aquaporins, which facilitate water movement between cytoplasm and matrix. This review discusses different phases of mitochondrial swelling and summarizes the potential effects of mitochondrial swelling on cell function. potassium homeostasis; depolarization; mitochondrial swelling     

Osmotic swelling-provoked release of organic osmolytes in human intestinal epithelial cells

Human Intestine 407 cells respond to osmotic cell swelling by the activation of Cl(-)- and K(+)-selective ionic channels, as well as by stimulating an organic osmolyte release pathway readily permeable to taurine and phosphocholine. Unlike the activation of volume-regulated anion channels (VRAC), activation of the organic osmolyte release pathway shows a lag time of approximately 30-60 s, and its activity persists for at least 8-12 min. In contrast to VRAC activation, stimulation of organic osmolyte release did not require protein tyrosine phosphorylation, active p21(rho), or phosphatidylinositol 3-kinase activity and was insensitive to Cl(-) channel blockers. Treatment of the cells with putative organic anion transporter inhibitors reduced the release of taurine only partially or was found to be ineffective. The efflux was blocked by a subclass of organic cation transporter (OCT) inhibitors (cyanine-863 and decynium-22) but not by other OCT inhibitors (cimetidine, quinine, and verapamil). Brief treatment of the cells with phorbol esters potentiated the cell swelling-induced taurine efflux, whereas addition of the protein kinase C (PKC) inhibitor GF109203X largely inhibited the response, suggesting that PKC is involved. Increasing the level of intracellular Ca(2+) by using A-23187- or Ca(2+)-mobilizing hormones, however, did not affect the magnitude of the response. Taken together, the results indicate that the hypotonicity-induced efflux of organic osmolytes is independent of VRAC and involves a PKC-dependent step.     

Epidermal Growth Factor Receptor is a Common Element in the Signaling Pathways Activated by Cell Volume Changes in Isosmotic,Hyposmotic or Hyperosmotic Conditions

Changes in external osmolarity, including both hyper- or hyposmotic conditions, elicit the tyrosine phosphorylation of a number of tyrosine kinase receptors (TKR). We show here that the epidermal growth factor receptor (EGFR) is activated by both cell swelling (hyposmolarity, isosmotic urea, hyperosmotic sorbitol) or shrinkage (hyperosmotic NaCl or raffinose) and discuss the mechanisms by which these apparently opposed conditions come to the same effect, i.e., EGFR activation. Evidence suggests that this results from early activation of integrins, p38 and tyrosine kinases of the Src family, which are all activated in the two anisosmotic conditions. TKR transactivation by integrins and p38 is likely occurring via an effect on the metalloproteinases. Information discussed in this review, points to TKR as elements in osmotransduction as a useful mechanism to amplify and diversify the initial response to anisosmolarity and cell volume changes, due to their privileged situation as convergence point for numerous intracellular signaling pathways. The variety of effector pathways connected to TKR is advantageous for the cell to cope with the changes in cell volume including adaptation to stress, cytoskeleton remodeling, adhesion reactions, cell survival and the adaptive mechanisms to ultimately restore the original cell volume. Special Issue dedicated to Dr. Simo S. Oja     

Ionomycin produces an improved volume recovery by an increased efflux of taurine from hypoosmotically stressed molluscan red blood cells.

Nucleated erythrocytes of the blood clam, Noetia ponderosa, recover cell volume after a hypoosmotic stress by an efflux of K+, Cl- and taurine. When the cells are exposed to ionomycin followed by hypoosmotic stress, swelling is less and volume recovery is both faster and more complete than in control cells without the ionophore. The improved volume recovery is caused by a large increase in the efflux of taurine. The taurine efflux is altered by changing Ca2+ concentrations in the presence of the ionophore. Potassium regulation by the osmotically stressed erythrocytes is also increased in the presence of ionomycin, but only by a small amount, perhaps accounting for the initial decrease in swelling. Variation of Ca2+ in the presence of ionomycin without osmotic stress produces no change in the regulation of either osmolyte. These results indicate that both the osmotic stress and an increase in [Ca2+]i are required for the permeability change that produces taurine efflux.